Injectable composition for preventing hair loss or stimulating hair growth

ABSTRACT

The present invention relates to an injectable pharmaceutical composition containing keratin for preventing hair loss or stimulating hair growth, wherein the composition is excellent in hair follicle generating and hair growth stimulating effects, and thus can be favorably used as a preventive and therapeutic agent for hair loss or a stimulating agent for hair growth.

TECHNICAL FIELD

This application claims priority to and the benefit of Korean PatentApplication No. 10-2017-0014047 filed in the Korean IntellectualProperty Office on Jan. 31, 2017, the entire contents of which areincorporated herein by reference.

The present invention relates to an injectable composition containingkeratin for preventing hair loss or stimulating hair growth.

BACKGROUND ART

Hair loss refers to a state in which there is no hair in a place wherehair should be normally present, and genetic factors are known to be themain cause. Recently, due to environmental pollution, westernized eatinghabits such as instant foods, frequent perms and dyeing, and incorrectscalp cares as well as the increase of social stress, the population ofhair loss is gradually increasing. However, the definite cause of hairloss has not yet been clarified. Recently, more and more people aresuffering from alopecia, and the age group is lowering.

Even in the treatment of alopecia, there is still no clear effect, thereare several taking prescriptions related to hair loss, such asSineungyangjindan in Donguibogam, and there are methods of massagingsesame oil (sesame) in a hair loss area, applying a specific herbalmedicine prescription by primary precipitate, and stimulating a specificacupoint, but it is known that there are many individual differences inthe effect. In addition, there is still no report of a new drugsubstance that can be generally applied.

As a method for treating alopecia in the related art, there is apreparation using female hormone as a main material in relation tohormonal theory, but there is a report of occurrence of skininflammation and adverse effects due to administration of hormone, andits use is being stopped at present. As a representative hair growthagent currently in use, there are minoxidil(6-amino-1,2-dihydro-1-hydroxy-2-imino-4-phenoxypyrimidine) in U.S. Pat.No. 3,382,247 and finasteride from Merck mentioned in U.S. Pat. No.5,215,894, which have been first developed and used for stimulatingblood circulation, have been known to patients using the hair growthagent to have a hair growth effect as side effects, and then have beenapproved by the US Food and Drug Administration (FDA) as a raw materialfor hair growth to be used as a hair growth treating agent.

However, the minoxidil has been reported to have a sticky feel and sideeffects that cause irritation to the skin, and the finasteride has beencurrently used as a preparation for oral administration, but adverseeffects such as sexual dysfunction have been reported depending on itsconsumption, and there is a side effect to be taken steadily for hairloss.

Thus, it is necessary to develop a new therapeutic agent that canreplace the existing hair growth agent and has an excellent effect ofpreventing hair loss and stimulating hair growth.

PRIOR ART DOCUMENTS Patent Document

(Patent Document 1) U.S. Pat. No. 3,382,247

(Patent Document 2) U.S. Pat. No. 5,215,894

DISCLOSURE Technical Problem

In order to solve the problems, the present inventors discovered thatkeratin may stimulate hair growth or prevent hair loss while continuinga research about a new therapeutic agent that can replace existing hairgrowth agent. Furthermore the present inventors discovered that when thekeratin was injected into the skin tissue, it was more effective instimulating hair growth or preventing hair loss than when the keratinwas applied to skin, and thus the inventors completed the presentinvention.

Therefore, an object of the present invention is to provide aninjectable pharmaceutical composition containing keratin for preventinghair loss or stimulating hair growth.

Technical Solution

In order to achieve the object, according to one embodiment of thepresent invention, an injectable pharmaceutical composition is providedwhich contains keratin that prevents hair loss or stimulates hairgrowth.

In one embodiment of the present invention, the keratin may behydrolyzed keratin or keratin linked to a water-soluble polymer.

The hydrolyzed keratin may have a molecular weight of 500 to 10,000Daltons.

The water-soluble polymer may be selected at least one from the groupconsisting of hyaluronic acid, polyethylene glycol (PEG), alginic acid,pectin, carrageenan, chondroitin sulfate, dextran sulfate, chitosan,polylysine, collagen, gelatin, carboxymethyl chitin, fibrin, dextran,agarose, pullulan, polyacrylamide (PAAm), poly(N-isopropylacrylamide)(P(NIPAAm-co-AAc)), poly(N-isopropylacrylamide-co-ethylmethacrylate)(P(NIPAAm-co-EMA)), polyvinyl acetate/polyvinyl alcohol (PVAc/PVA),poly(N-vinylpyrrolidone) (PVP), poly(methyl methacrylate-co-hydroxyethylmethacrylate) (P(MMA-co-HEMA)), poly(polyethyleneglycol-co-peptide(P(PEG-co-peptide)), alginate-g-(polyethylene oxide-polypropyleneoxide-polyethylene oxide) (alginate-g-(PEOPPO-PEO)), poly(polylacticacid-co-glycolic acid)-co-serine) (P(PLGA-co-serine)), collagenacrylate,alginate-acrylate, poly(hydroxypropyl methacrylamide-g-peptide) (P(HPMA-g-peptide)), poly(hydroxyethyl methacrylate/matrigel)(P(HEMA/Matrigel)), hyaluronic acid-g-N-isopropylacrylamide(HA-g-NIPAAm), polyethylene oxide (PEO), a polyethyleneoxide-polypropylene oxide copolymer (PEO-PPO, Pluronic series), apolyethylene oxide-polylactic acid copolymer (PEO-PLA), a polyethyleneoxide-polylactic glycolic acid copolymer (PEO-PLGA), a polyethyleneoxide-polycaprolactone copolymer (PEO-PCL), polyoxyethylene alkyl ethers(Brij Series), polyoxyethylene castor oil derivatives (Cremophores),polyoxyethylene sorbitan fatty acid esters (Tween Series), andpolyoxyethylene stearates.

The keratin may stimulate hair regeneration.

The pharmaceutical composition may increase an expression level of atleast one selected from the group consisting of beta-catenin, Sox-9,Sox-2, alkaline phosphatase, CD133, FGF7, FGF10, BMP6, P-cadherin,E-cadherin, MSX2, FOXN1 and CD10.

The pharmaceutical composition may be administered to the dermal layeror the subcutaneous tissue.

The pharmaceutical composition may further include a penetrationenhancer.

The penetration enhancer may be selected at least one from the groupconsisting of sulphoxide, azone, pyrrolidone, fatty acids, loweralcohols having 1 to 4 carbon atoms, higher fatty alcohols having 6 ormore carbon atoms, glycols, urea, terpene, terpenoid, and phospholipid.

The hydrolyzed keratin may be prepared by a preparation method ofhydrolyzed keratin, including S1) reacting keratin with hydrolase; andS2) removing the hydrolase.

The hydrolase may be proteinase-K.

The hydrolase may be immobilized on beads.

The preparation method of hydrolyzed keratin may further include S3)removing the activity of the hydrolase after step S2).

Advantageous Effects

The injectable pharmaceutical composition containing keratin of thepresent invention stimulates hair growth and formation of hairfollicles.

Furthermore, when the injectable pharmaceutical composition containingkeratin of the present invention is injected into the skin tissue, it ismore effective to stimulate hair growth or prevent hair loss than whenthe pharmaceutical composition is applied to the skin.

Therefore, the pharmaceutical composition of the present invention canbe used as a therapeutic agent for preventing hair loss or stimulatinghair growth.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates data obtained by NMR analysis of PEGylated keratin.

FIG. 2 is a diagram illustrating a hair growth effect, 2 weeks afterinjecting Examples 1 to 6 into hair-removed mice.

FIG. 3 is a photograph illustrating the degree of hair follicleformation by injecting Examples 1 to 6 into hair-removed mice and thenstaining the injected mice with hematoxylin-eosin after 2 weeks.

FIG. 4 is a graph illustrating the number of hair follicles formed ineach stage of hair growth, 2 weeks after injecting Examples 1 to 6 intohair-removed mice.

FIG. 5 is a graph illustrating sizes of hair follicles formed, 2 weeksafter injecting Examples 1 to 6 into hair-removed mice.

FIG. 6 is a photograph of a result of SDS-PAGE analysis for confirmingthat hydrolyzed keratin having a different molecular weight from naturalkeratin may be prepared.

FIG. 7 is a diagram of confirming that cell proliferation is inhibitedwhen human dermal papilla cells are treated with keratin.

FIG. 8 is a photograph of confirming that keratin is attached to thecell surface by analyzing interaction with cells and then formation ofcell aggregates is induced by keratin, when human dermal papilla cellsare treated with keratin.

FIG. 9 is a diagram illustrating that expression of mRNA associated withcell proliferation is suppressed and expression of mRNA associated withcell migration, induction of cell aggregates, and synthesis of anextracellular matrix is increased, when human dermal papilla cells aretreated with keratin.

FIG. 10 is a diagram illustrating that molecular expression of FGF7,FGF10 and BMP6, which are known as factors inducing hair growth, isincreased when human dermal papilla cells are treated with keratin.

FIG. 11 is a graph of confirming that expression of beta-catenin andP-cadherin as factors related with cell migration and differentiationinto matrix cells is increased and expression of CD34 as a molecule thatreduces expression at differentiation is decreased, when human outerroot sheath cells are treated with keratin.

FIG. 12 is a graph of confirming that gene expression of MSX2, FOXN1,and CD10 as factors related with differentiation into matrix cells isincreased and gene expression of KRT5 and ITGA6 as molecules withreduced expression at differentiation is decreased, when human outerroot sheath cells are treated with keratin.

FIG. 13 is a diagram of confirming that expression of a beta-cateninmolecule as a factor related with cell migration and activity of cloneand a Sox-9 molecule involved in stemness is increased, when outer rootsheath cells are treated with hydrolyzed keratin.

FIG. 14 is a graph illustrating that expression of a Sox-9 gene and abeta-catenin gene involved with stemness is increased when outer rootsheath cells are treated with hydrolyzed keratin.

FIG. 15 is a graph of confirming that a beta-catenin molecule isexpressed four times or more when outer root sheath cells are treatedwith hydrolyzed keratin.

FIG. 16 is a diagram of confirming that formation of cell aggregatesinvolved with hair regeneration and stemness is increased five times ormore when human dermal papilla cells are treated with hydrolyzedkeratin.

FIG. 17 is a photograph of confirming that activity of alkalinephosphatase by which alkaline phosphate cells are expressed is increasedwhen human dermal papilla cells are treated with hydrolyzed keratin.

FIG. 18 is a photograph of confirming that expression of beta-catenin,Sox-2, alkaline phosphatase (ALPase), and CD133, FGF-7 and FGF-10molecules as factors of hair regeneration and stemness is increased whenhuman dermal papilla cells are treated with hydrolyzed keratin.

MODE FOR INVENTION

The present invention provides an injectable pharmaceutical compositioncontaining keratin for preventing hair loss or stimulating hair growth.

The keratin may be a true keratin constituting hair, fleece, feather,horn, nail, horseshoe, or the like, and may be keratin existing in theskin, nervous tissue or the like.

In addition, the keratin includes amino acids such as glutamic acid,arginine, and cystine, and particularly may contain a high content ofcystine.

The keratin may be any commercially available keratin, processed to besoluble in water or a water-soluble solvent, preferably, humanhair-derived keratin, and the molecular weight thereof may be 40,000 to70,000 Daltons.

The keratin may be hydrolyzed keratin or keratin linked to awater-soluble polymer.

The hydrolyzed keratin is a protein that exhibits water solubility bydecomposing insoluble natural keratin while retaining the properties ofkeratin, and may be used in combination with keratin-derived peptides.The hydrolyzed keratin may be prepared by hydrolyzing keratin with acid,alkali, oxygen, or hydrolase, but is not limited thereto, and may beobtained by a general method of preparing a hydrolyzed keratin having amolecular weight of 500 to 10,000 Daltons from natural keratin.

The hydrolyzed keratin may be prepared by a preparation method ofhydrolyzed keratin, including S1) reacting keratin with a hydrolase; andS2) removing the hydrolase.

The step S1) is a step of preparing keratin hydrolyzed from keratinusing the hydrolase.

Here, the hydrolase is classified into 9 groups according to theiraction targets, as groups classified into enzymes that catalyzehydrolysis reactions when enzymes are classified systematically.

The hydrolase is an enzyme which may hydrolyze natural keratin becausethe keratin is a protein, and preferably an enzyme that acts on peptidebonds. Specifically, the enzyme that acts on the peptide bonds may beany one selected from proteases including Leucyl aminopeptidase,carboxypeptidase, pepsin, trypsin, and chymotrypsin. More preferably,the enzyme is a proteinase-K, but is not limited thereto.

In the method for preparing the hydrolyzed keratin, if the step S) isabsent or is not properly performed, the yield of the hydrolyzed keratinexhibiting an effect of preventing hair loss or stimulating hair growthmay be insufficient.

Next, the step S2) is a step of obtaining only the hydrolyzed keratinfrom a mixture of the hydrolase and the hydrolyzed keratin. Thehydrolase may be immobilized on beads. The hydrolase may be separatedfrom the hydrolyzed keratin according to the physical/chemicalproperties of the bead using a difference in magnetism of the bead, adifference in weight, a difference in adsorption, or the like.

The beads may be selected at least one from the group consisting ofpoly-L-lactic acid (PLLA), a poly-lactic acid-glycolic acid copolymer(PLGA), polyglycolic acid (PGA), polyurethane (PU),polymethylmethacrylate (PMMA), polyethylene (PE), and ferromagnetite,but the present invention is not limited thereto. Specifically, thebeads may be agarose beads.

In the method for preparing the hydrolyzed keratin, if the step S2) isabsent or is not properly performed, the purity of the hydrolyzedkeratin exhibiting an effect of preventing hair loss or stimulating hairgrowth may be lowered.

In addition, the method for preparing the hydrolyzed keratin may furtherinclude S3) removing the activity of the hydrolase, after the step S2).

The step S3) is for stopping the hydrolysis reaction of the keratin byremoving the activity of the hydrolase, and may be left at a hightemperature for several hours so that the hydrolase does not react withthe keratin.

In the method for preparing the hydrolyzed keratin, if the step S3) isincluded, the purity of the hydrolyzed keratin may be increased and thehydrolyzed keratin having a uniform molecular weight may be obtained.

For example, the hydrolyzed keratin may be obtained by mixing human hairwith a mixture of chloroform and methanol in a ratio of 2:1 to removelipid, stirring the mixture in a 2% peracetic acid solution for 12hours, and then adding 5% 2-mercaptoethanol, 5 M urea, 2.6 M thiourea,and 25 mM Tris-HCl (pH 8.5), and reacting at 50 to 72 hours, but thepresent invention is not limited thereto.

In the present invention, the water-soluble polymer may be selected atleast one from the group consisting of hyaluronic acid, polyethyleneglycol (PEG), alginic acid, pectin, carrageenan, chondroitin sulfate,dextran sulfate, chitosan, polylysine, collagen, gelatin, carboxymethylchitin, fibrin, dextran, agarose, pullulan, polyacrylamide (PAAm),poly(N-isopropylacrylamide) (P(NIPAAm-co-AAc)),poly(N-isopropylacrylamide-co-ethylmethacrylate) (P(NIPAAm-co-EMA)),polyvinyl acetate/polyvinyl alcohol (PVAc/PVA), poly(N-vinylpyrrolidone)(PVP), poly(methyl methacrylate-co-hydroxyethyl methacrylate)(P(MMA-co-HEMA)), poly(polyethyleneglycol-co-peptide(P(PEG-co-peptide)), alginate-g-(polyethylene oxide-polypropyleneoxide-polyethylene oxide) (alginate-g-(PEOPPO-PEO)), poly(polylacticacid-co-glycolic acid)-co-serine) (P(PLGA-co-serine)), collagenacrylate,alginate-acrylate, poly(hydroxypropyl methacrylamide-g-peptide) (P(HPMA-g-peptide)), poly(hydroxyethyl methacrylate/matrigel)(P(HEMA/Matrigel)), hyaluronic acid-g-N-isopropylacrylamide(HA-g-NIPAAm), polyethylene oxide (PEO), a polyethyleneoxide-polypropylene oxide copolymer (PEO-PPO, Pluronic series), apolyethylene oxide-polylactic acid copolymer (PEO-PLA), a polyethyleneoxide-polylactic glycolic acid copolymer (PEO-PLGA), a polyethyleneoxide-polycaprolactone copolymer (PEO-PCL), polyoxyethylene alkyl ethers(Brij Series), polyoxyethylene castor oil derivatives (Cremophores),polyoxyethylene sorbitan fatty acid esters (Tween Series), andpolyoxyethylene stearates. Preferably, the water-soluble polymer may bepolyethylene glycol or hyaluronic acid, but the present invention is notlimited thereto.

In the present invention, the polyethylene glycol may be, for example,O-methyl-O′-succinylpolyethylene glycol, dibasic acid polyethyleneglycol, α,ω-bis(2-carboxyethyl-polyethylene glycol,O,O′-bis(2-bromoethyl) polyethylene glycol, O,O′-bis(2-chloroethyl)polyethylene glycol, polyethylene glycol dimethylate,methoxypolyethylene glycol acetic acid,O-[2-(3-succinylamino)ethyl]-O′-methyl-polyethylene glycol,O-(2-bromoethyl)-O′-methylpolyethylene glycol,O-(2-chloroethyl)-O′-methylpolyethylene glycol, polyethylene glycolmonomethyl ether mesylate, aldehyde-functionalized polyethylene glycol,glycidyl ether functionalized polyethylene glycol, nitrophenyl carbonatefunctionalized polyethylene glycol, mesyl functionalized polyethyleneglycol, or tosyl functionalized polyethylene glycol, and preferablyO-methyl-O′-succinylpolyethylene glycol.

The molecular weight of the water-soluble polymer may be 1000 Da to 4000kDa, preferably 2000 Da to 3000 kDa. As a preferable example, hyaluronicacid of 1000 kDa to 4000 kDa or polyethylene glycol of 1000 Da to 20000Da may be used.

The keratin may stimulate hair regeneration.

Specifically, the keratin may induce the growth of human outer rootsheath cells or dermal papilla cells and the aggregation of cells tostimulate hair regeneration.

The “stimulation of hair growth” mentioned in the present inventionmeans stimulating “hair growth” and/or “hair regeneration”, the “hairregeneration” means inducing growth of human outer root sheath cells ordermal papilla cells or cell aggregation to ultimately prevent hair fromfalling out easily, and means inducing the formation of hair folliclesby stimulating formation of hair follicles at an anagen stage which isan initial stage of hair growth to regenerate the hair. Furthermore, the“stimulation of hair growth” includes increasing expression factorsinvolved to migration of human outer root sheath cells and activity ofclones or stemness.

In addition, the pharmaceutical composition according to the presentinvention may increase the expression levels of genes for inducing hairregeneration and hair growth contained in human outer root sheath cellsor dermal papilla cells.

The gene may be selected at least one from the group consisting ofbeta-catenin, Sox-9, Sox-2, alkaline phosphatase, CD133, FGF7, FGF10,BMP6, P-cadherin, E-cadherin, MSX2, FOXN1 and CD10.

The beta-catenin means a signaling substance that receives a signal of aWnt protein to migrate into the cell nucleus and regulates theexpression of a target gene involved in cell proliferation anddifferentiation, a substance which is contained in stem cells to begrown to hair, and a substance related with cell migration and clonalactivity.

The Sox-9 is a substance involved in stemness and means a gene expressedin the nucleus of outer root sheath (ORS) and sebaceous gland cells ofhair.

The Sox-2 means a substance which is highly expressed in embryonic stemcells, plays an important role not only in maintaining the pluripotencyof embryonic stem cells but also in making inducing pluripotent stemcells, and has an important function for maintaining stem cellcharacteristics.

The alkaline phosphatase as a kind of enzyme is a substance which existsin various part of body as several isoenzymes, a substance involved inangiogenesis in a hair matrix during hair growth and means a substanceof which activity is increased when hair growth occurs.

The CD133 is known as a protein which exists in the extracellularmembrane and is induced to be expressed when the cells grow, correspondsto a cluster of differentiation (CD)-based protein, and means asubstance which may have immunological properties of hair follicle stemcells.

The FGF7 is a keratinocyte growth factor and means a substance thatpenetrates into the dermis below the epidermis to strongly stimulate thegrowth and proliferation of keratinocytes.

The FGF10 is a fibroblast growth factor and means a substance thatpenetrates into the dermis below the epidermis to strongly stimulate thegrowth and proliferation of fibroblasts.

The BMP6 is a protein that regulates biological activity in variouskinds of cells including nerve cells and means a factor for inducing theformation of aggregates of human dermal papilla cells to induce hairgrowth.

The P-cadherin and E-cadherin as cadgerin-based proteins mean proteinswhich are used to attach cells, are linked to the actin cytoskeleton tohave a function of help in assembling the proteins and acts as signalingmolecules that change gene expression in cells, and mean factorsinvolved in migration of human outer root sheath cells anddifferentiation into matrix cells.

The MSX2, FOXN1, and CD10 mean factors involved in the differentiationof human outer root sheath cells into matrix cells.

The pharmaceutical composition may increase beta-catenin or Sox-9 inhuman outer root sheath cells to induce migration of outer root sheathcells and activity of clones.

The pharmaceutical composition may stimulate the formation of cellaggregates of human dermal papilla cells and increase the formation ofcell aggregates to induce hair growth and improve a hair regenerationeffect.

The pharmaceutical composition may increase the expression ofbeta-catenin, Sox-2, alkaline phosphatase, CD133, FGF7, and FGF10 inhuman dermal papilla cells, thereby increasing the stemness of humandermal papilla cells.

In one specific embodiment of the present invention, the hair of thedorsal part of the elderly mouse with reduced physical functions wasshaved and then a keratin solution was injected into the dorsal part toanalyze the density of the growing hair and the formation of hairfollicles. As a result, it was confirmed that the keratin stimulated theformation of hair follicles and hair regeneration. Thus, it has beenseen that the keratin may be used in the injectable pharmaceuticalcomposition for preventing hair loss or stimulating hair growth.

In the present invention, the term “injection” refers to the injectionof a drug solution intradermally, subcutaneously, intramuscularly orintraarterially using a syringe. Specifically, since the pharmaceuticalcomposition of the present invention has the effect of preventing hairloss or stimulating hair growth, the injection may be a subcutaneousinjection, but is not limited thereto. The syringe can be used not onlyas a general syringe capable of administering a drug solution through aneedle but also as a syringe used for subcutaneous administration of thepharmaceutical composition of the present invention.

In the present invention, the injectable pharmaceutical composition maybe in the form of a liquid or dry powder. The dry powder for injectionmay be reconstituted with at least one selected from the groupconsisting of water for injection, a physiological saline solution, aglucose solution, and an amino acid solution to be administered to asubject.

The amount of an active ingredient contained in the composition of thepresent invention depends on a condition of a subject to beadministered, a desired degree of treatment, and the like. Specifically,the keratin of the present invention may be contained in a concentrationof 0.001 to 10 (w/v) %, specifically 0.01 (w/v) % to 5 (w/v) %, and morespecifically 0.05 to 2 (w/v) % based on the injectable pharmaceuticalcomposition.

When the concentration of the keratin is less than 0.001 (w/v) %, theeffect of preventing hair loss or stimulating hair growth may beinsufficient, and if the concentration of keratin is more than 10 (w/v)%, there is a problem that side effects in the body may be shown.

In the present invention, the composition may further include apenetration enhancer.

The pharmaceutical composition may be administered locally to a hairloss site or a site where hair growth is to be stimulated, preferablymay be administered to the dermal layer or subcutaneous tissue. At thistime, the dermal layer is the thickest layer below the epidermis, whichmeans a layer where a vascular system, a nervous system, a lymphaticsystem, etc. are intricately intertwined, hair follicles are generatedand grow, and hair follicles are contained.

The subcutaneous tissue is a portion between the muscle and the bonebelow the dermal layer, which has fat cells containing a large amount offat to give the softness to the body softness, have the contour, and beused as energy. It also means that arteries and lymph are circulating.

The penetration enhancer may be at least one selected from the groupconsisting of sulphoxide, azone, pyrrolidone, fatty acids, loweralcohols having 1 to 4 carbon atoms, higher fatty alcohols having 6 ormore carbon atoms, glycols, urea, terpene, terpenoid, and phospholipid.However, the penetration enhancer is not limited thereto and may includegeneral stimulators that are used in the injectable pharmaceuticalcomposition to stimulate transdermal penetration of an activeingredient.

The sulphoxide may be at least one selected from the group consisting ofdimethyl sulfoxide (DMSO), dimethyl acetamide (DMAC), dimethyl formamide(DMF), and decylmethyl sulfoxide (DCMS), but is not limited thereto.

The pyrrolidione may be at least one selected from the group consistingof N-methyl-2-pyrrolidone (NMP) and 2-pyrrolidone (2P) but is notlimited thereto.

The azone may be 1-dodecylazacycloheptan-2-one or laurocapram.

The penetration enhancer may be contained in a generally acceptableamount to the injectable composition depending on a kind to be used.

The pharmaceutical composition of the present invention may be usedalone or in combination with hormonal therapy, chemotherapy, and methodsusing a biological response modifier for preventing hair loss orstimulating hair growth.

The present invention provides a method for preventing hair loss orstimulating hair growth including preparing an injectable pharmaceuticalcomposition containing keratin; and administering the injectablepharmaceutical composition to a subject.

The “administration” means introducing a desired substance into thesubject in an appropriate manner. In the present invention, thecomposition may be administered to the dermal layer or the subcutaneoustissue because of its characteristic of preventing hair loss orstimulating hair growth by injecting into the subject.

The pharmaceutical composition of the present invention varies dependingon various factors including the activity of a specific compound used,age, weight, general health, gender, diet, time of administration, routeof administration, rate of excretion, drug combination, and the severityof a specific disease to be prevented or treated. The dose of thepharmaceutical composition may vary depending on a condition of thepatient, a body weight, the degree of disease, a type of drug, and routeand period of administration, but may be suitably selected by thoseskilled in the art and may be administered in an amount of 0.0001 to 100mg/kg or 0.001 to 100 mg/kg. The administration may be performed once aday or several times a day. The dose does not limit the scope of thepresent invention in any aspect. In addition, the dose and concentrationmay vary depending on the human hair growth area, and the concentrationof the composition is preferably 0.1 to 100 mg/ml but is not limitedthereto.

The “subject” means all animals such as mice, rats, and livestock,including humans that have or may develop hair loss symptoms.Specifically, the subject may be mammals including humans.

Hereinafter, preferred Preparation Examples or Experimental Examples arepresented in order to assist understanding of the present invention.However, the following Preparation Examples or Experimental Examples arejust provided to more easily understand the present invention andcontents of the present invention are not limited by the embodiment.

Preparation Example 1: Obtainment of Keratin

Human hair was cleanly washed with a weak detergent and rinsed severaltimes with distilled water. For lipid removal, the hair was placed in abeaker and added with a mixture of chloroform and methanol at a ratio of2:1 until the hair sank. After 24 hours, the solution used for lipidremoval was washed several times with distilled water until the solutionwas not left in the hair, and then the hair was air-dried. 20 g of thehair which had been air-dried was added in 800 mL of 2% acetic acid(Sigma aldrich), and the mixture was stirred at 37° C. for 12 hours at300 rpm. After 12 hours, the hair was sieved and washed with distilledwater to remove remaining oxides. The hair was added in a 400 mL Shindaisolution (5% 2-mercaptoethanol, 5 M of urea, 2.6 M of thiourea (Sigmaaldrich) and 25 mM Tris-HCl (pH 8.5)) and reacted for 72 hours underconditions of 50° C. and 400 rpm. Thereafter, the hair solution wasadded in a 50 mL tube and centrifuged at 3500 rpm for 20 minutes. Thesupernatant was collected and dialyzed using a 12-14 kDa cut-offSpectra/Por® 4 dialysis membrane (Spectrum). The dialyzed keratin liquidsample was lyophilized to prepare a keratin powder.

Preparation Example 2: Preparation of Hydrolyzed Keratin

First, dH₂O was added to a brown vial (10 ml, 22*48) made ofpolyethylene terephthalate having a screw cap and 20 mg of Tritirachiumalbum-derived proteinase K (immobilized to Eupergit, Sigma-Aldrich) wasadded and dissolved at a concentration of 2 mg/ml. The proteinase-K wasfully dissolved and then 400 mg of the keratin powder prepared accordingto Preparation Example 1 was weighed using an electronic balance formeasurement of fine part unit count (0.001 g to 620 g, AJ-620E) andadded into the vial. The vial was placed in a digital drying oven andthen stirred at 37° C. at rate of 300 rpm for 1 hour using an egg-white,5×2 mm magnetic stir bar (BOOC3ME4ZA, 1572500 IKAFLON 40, IKA) and amobile MS-H-S10 10-Channel magnetic stirrer. The fully hydrolyzedkeratin solution was placed in a centrifuge and operated at 250 g for 10min to precipitate Proteinase-K immobilized Eupergit C and then theprecipitate was removed. Only the supernatant was obtained and was addedto an Eppendorf® PCR tube by 10 μl. The tube was boiled for 1 hour at99° C. in CFX96 Touch™ Real-Time PCR detection system equipment toremove the activity of the proteinase-K. The hydrolyzed keratin liquidsample was lyophilized to prepare a hydrolyzed keratin powder.

Preparation Example 3: Preparation of PEGylated Keratin PreparationExample 3-1: Synthesis of PEGylated Keratin

First, 0.5 g of the keratin powder obtained in Preparation Example 1 wasweighed using an electronic balance (0.001 g to 620 g, AJ-620E) formeasurement of fine part unit count and placed in a 500 mL Pyrex beaker(ISOLAB, YLS, Germany). 100 mL of tertiary water was added to a beakerand stirred at a rate of 300 rpm for 24 hours using a 40×8 mm magneticstir bar (BOOC3ME4ZA, 1572500 IKAFLON 40, IKA) and an MS-H-S1010-Channel magnetic stirrer.

300 mg of O-methyl-O′-succinyl polyethylene glycol 5000 (mPEG,17929-5G-F, Lot #R063737/2V Sigma aldrich) modified withmethoxypolyethylene glycol was dissolved in 20 mL of tertiary water for1 hour. 16 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methoxymorpholinium chloride (DMTMM) n-hydrate (327-53752, wako chemical) wasadded to an mPEG solution and then stirred for 1 hour. The keratinsolution and a binding solution of mPEG and DMTMM were placed in a 500mL beaker and stirred at a rate of 500 rpm at room temperature for 3days using a 40×8 mm magnetic stir bar (1572500 IKAFLON 40, IKA) and anMS-H-S10 10-Channel magnetic stirrer.

Preparation Example 3-2: Purification of PEGylated Keratin

After the reaction of Preparation Example 3-1 was completed, thePEGylated keratin solution was added to a Spectra/Por permeable membranetube (diameter of 16 mm, 25 mm of flat width, MW10000, Spectrum) by 100mL per tube and packed using a Spectra/Por RC dialysis tubing closure(maximum width of 55 mm, Orange, Spectrum). A 5 L plastic beaker (197 mmin diameter, 265 mm in height) was filled with 4 L of tertiary water,added with the tube containing the PEGylated keratin solution, addedwith a magnetic bar, and stirred to perform dialysis. At this time, newtertiary water was changed every 12 hours and the dialysis was performedwhile the tertiary water was changing a total of 6 times at roomtemperature for 3 days. The final PEGylated keratin solution was placedin a 50 mL centrifuge tube (17×120 mm, BD Falcon) to be divided by 40mL, and then completely frozen at −70° C. for 24 hours in a cryogenicrefrigerator (Nihon freezer, Upright type, Single cooling type, 5421).The lid of the centrifuge tube was opened and the PEGylated keratinsolution was placed in a freeze dryer (ALPHA 2-4 LSC PLUS, Laboratoryfreeze dryers, Christ) and dried for 3 days in a vacuum state at −85° C.to be completely powdered. Next, the PEGylated keratin was analyzed byNMR. As a result, as illustrated in FIG. 1, it was confirmed that theNMR spectra of the PEGylated keratin had signature peaks for keratin andPEG,

Preparation Example 3-3: Preparation of PEGylated Keratin SolutionContaining Hydrogen Peroxide

20 mg of the PEGylated keratin obtained in Preparation Example 3-2 wasmeasured using an electronic balance ((0.001 g to 620 g) AJ-620E) formeasurement of fine part unit count. 102 μl of H₂O₂ (hydrogen peroxide30%, molecular weight 34.01, JUNSEI CHEMICAL, 7722-84-1) was added to898 μl of tertiary distilled water to make 1 mL of 1 M concentration,and then serial dilution was performed with distilled water 100 timesand 20 times to prepare a final 500 uM of H₂O₂ solution. The prepared500 uM of H₂O₂ solution was added with 20 mg of the PEGylated keratinand vortexed for 5 minutes to prepare a PEGylated keratin solutioncontaining 500 uM H₂O₂. A glass bottle containing the prepared solutionwas sealed with a nylon vacuum packing machine 150×200 mm (NY/PELLDPE)(80 um) using a pneumatic vacuum packing machine and then stored.

Experimental Example 1: Hair Growth Effect in Animal ExperimentExperimental Example 1-1: Preparation of Animal Model

Twenty-four C57BL/6J aged mice (Charles River Corp. Inc., Barcelona,Spain) weighing 20 to 25 g, aged 12 weeks, were used as an animal modelto verify a hair growth effect of keratin. After the mice wereanesthetized with isoflurane, the dorsal parts of the mice were shavedwith a clipper, and the hair and root were shaved while leaving atelogen stage hair root. The skin was disinfected with a povidone-iodinesolution and then wiped with 60% alcohol.

Experimental Example 1-2: Hair Growth Effect of Keratin

The dorsal parts of the mice prepared according to Experimental Example1-1 were administered and prepared as illustrated in Table 1 below. Anormal control group was a mouse that had not been subjected to anytreatment after hair cutting. All mice were bred for 2 weeks, and at 2weeks, hair cut areas were observed with a stereomicroscope (SZX16,Olympus). The results were illustrated in FIG. 2.

TABLE 1 Classification Ingredient Administration method Normal — —control group Positive 3% minoxidil Apply on the skin control daily for2 weeks group (application amount 100 μl/cm²) Example 1 0.5 w/v % ofkeratin- Subcutaneous single containing solution injection (300 μl)obtained from Preparation Example 1 Example 2 1 w/v % of keratin-Subcutaneous single containing solution injection (300 μl) obtained fromPreparation Example 1 Example 3 0.5 w/v % of hydrolyzed Subcutaneoussingle keratin-containing injection (300 μl) solution obtained fromPreparation Example 2 Example 4 1 w/v % of hydrolyzed Subcutaneoussingle keratin-containing injection (300 μl) solution obtained fromPreparation Example 2 Example 5 0.5 w/v % of PEGylated Subcutaneoussingle keratin-containing injection (300 μl) solution obtained fromPreparation Example 3-2 Example 6 0.5 w/v % of PEGylated Subcutaneoussingle keratin-containing injection (300 μl) solution obtained fromPreparation Example 3-2 Comparative 1 w/v % of keratin-containing Applyon the skin Example 1 solution obtained from (application amountPreparation Example 1 100 μl/cm²)

As a result, it was shown that in Examples 1 to 6, a hair growth effectwas remarkably superior to that in the normal control group, and inExamples 1 to 6, hair was uniformly grown and in Examples 1, 2 and 4,the hair was very densely grown. Accordingly, it was confirmed that whenthe keratin was subcutaneously injected, the hair growth effect wasbetter than that when the keratin was applied on the skin, it wasconfirmed that the injectable pharmaceutical composition containingkeratin according to the present invention had effects of preventinghair loss and stimulating hair growth, and it was confirmed that theeffects of preventing hair loss and stimulating hair growth were fast.

In addition, the injectable pharmaceutical composition containing thekeratin of the present invention had a remarkably high effect ofpreventing hair loss and stimulating hair growth than when a formulationwas applied to the skin as a conventional external preparation for skin,and the subcutaneous injection using the formulation as an injectableproduct had a higher bioavailability.

On the other hand, when the formulation was converted into an injectionother than the external preparation for skin, side effects such astoxicity were also increased due to an increase in bioavailability whenused as an injection as compared with when used as an externalpreparation for skin, and thus it is also important to solve the sideeffects. Therefore, it was confirmed through FIG. 2 that the injectablepharmaceutical composition containing the keratin of the presentinvention had no toxicity at the time of subcutaneous injection of theinjectable pharmaceutical composition containing keratin, and aftersubcutaneous injection of the pharmaceutical composition, there were noside effects that occurred by reacting with blood or moisture in thebody.

Experimental Example 1-3: Histological Analysis of Hair Growth Effect ofKeratin

After completion of Experimental Example 1-2, tissue specimens wereobtained by sacrificing the mice to which the solutions of Examples 1 to6 were administered, a normal control and a positive control,respectively. The tissue specimens were stained with hematoxylin andeosin (H&E), observed, and analyzed and the results are illustrated inFIGS. 3 to 5.

As a result, it was confirmed that formation of hair follicles occurredin mice administered with the keratin solutions of Examples 1 to 6 at amuch higher frequency than the normal control (FIG. 3). In particular,it was confirmed that in the mice administered with the keratin andhydrolyzed keratin solutions of Examples 1 to 4, the hair follicle sizewas increased as compared with the mice to which the PEGylated keratinsolutions of Examples 5 to 6 was administered. Specifically, asillustrated in FIG. 4, as a result of the analysis of each stage of hairgrowth, it was confirmed that the formation of hair follicles wasstimulated in an anagen stage, which is an early stage of hair growth,and as a result, formation of new hair follicles was induced (FIG. 4).In addition, as the concentration of the keratin solution administeredto the mice increased, the diameter of hair follicles tended to increase(FIG. 5).

Experimental Example 2: Identification of Hydrolyzed Keratin

Whether the hydrolyzed keratin was prepared according to PreparationExample 2 was identified by SDS-PAGE and illustrated in FIG. 6.

Specifically, a SeeBlue® plus 2 Pre-Stained Standard (novex) size markerwas taken out at room temperature and the solution was vortexed to becompletely mixed. A total of 40 μl of a sample obtained by mixing 10 μlof Bolt™ LDS Sample Buffer (4×), 4 μl of Bolt™ LDS Reducing Agent (10×),and 26 μl of 4% hydrolyzed keratin was vortexed. A hydrolyzed keratinsample was prepared by setting the temperature of a heating block(HYSC_Introduction_Heating Block_2014) at 100° C. and heating for 10minutes. After a comb portion of a Bolt Bis-Tris 4-12% gradient mini gel(Invitrogen) was removed, wells were washed with a 1×MES running bufferand a cover tape at the end of a cassette was removed. Before loadingthe sample, a chamber of the cassette was filled with the 1×MES runningbuffer and locked with a cassette clamp to prevent the buffer from beingremoved. The wells to which the sample was to be loaded were filled withthe 1×MES running buffer. 30 μl of a 4% hydrolyzed keratin sample and 10μl of a SeeBlue® plus2 Pre-Stained Standard (novex) size marker wereloaded, respectively. 0.012, 0.015, and 0.020 mg/600 μl PAA keratinsamples were used as a control. The cassette was filled with the 1×MESrunning buffer by an outer scale, loaded with the size marker and the 4%hydrolyzed keratin sample, and then run at 165V for 40 minutes with aPowerPac™ Basic Power Supply (Bio-rad). After running, the cassetteclamp was released and only the loaded gel was obtained and stained withComassie brilliant blue G-250 (Sigma) for 24 hours. The size marker andthe protein were identified after washing with a destain solution (10%(v/v) acetic acid, 40% (v/v) methanol) until the band was visible.

As a result, as illustrated in FIG. 6, it was confirmed that in the caseof natural keratin extracted from the SDS-PAGE analysis result, aprotein band was shown at a molecular weight level of about 50 kDaltons, but in the case of hydrolyzed keratin, a band was smearly shownat a molecular weight level of 3 to 6 k Daltons. As a result, it can beseen that the hydrolyzed keratin is constituted by various molecularweights of degraded peptides.

Experimental Example 3: Induction of Cell Aggregates and ExpressionInduction of Hair Growth-Related Factors of Keratin for Human DermalPapilla Cells In Vitro

In order to confirm a hair regeneration effect of keratin, expression offactors related to hair growth and induction of aggregation and hairgrowth was confirmed.

It was confirmed whether a keratin solution prepared using human dermalpapilla cells showed efficacy in cell proliferation.

Specifically, human dermal papilla cells were inoculated on a 24-wellplate by 2×10⁴ cells, and after 24 hours, the medium was replaced with aDMEM normal medium at a high concentration or a DMEM normal mediumcontaining 1% (w/v) of keratin at a high concentration. After 1, 3, and5 days of culture, cell proliferation assay was performed. After thewells were washed with DPBS, a Cell Counting Kit-8 solution (Dojindolaboratory) 10-fold diluted with the high-concentration DMEM normalmedium was added to each well, light was blocked with aluminum foil, andthen the cells were incubated for 1 hour 30 minutes at 37° C. Afterincubation, 100 μl of each well was transferred to a 96-well plate andabsorbance was measured at 450 nm wavelength (O.D 450 nm) using a96-well format plate reader (ELX 800 universal microplate reader,BioTek, Inc.). The results were illustrated in FIG. 7.

As a result, it was found that the proliferation of human dermal papillacells was inhibited during treatment of keratin (FIG. 7). In addition,after treatment of keratin complexed with a fluorescent substance, as aresult of analyzing interaction with cells, it was confirmed thatkeratin was adhered to the cell surface, and formation of cellaggregates according to cell migration was induced by keratin over time(FIG. 8).

To further confirm these results by mRNA expression analysis, first,human dermal papilla cells were inoculated in a 6-well late by 2×10⁵cells and after 24 hours, the medium was replaced with a DMEM normalmedium at a high concentration or a DMEM normal medium containing 1%(w/v) of keratin at a high concentration. After keratin treatment for 24hours, for RNA sequencing analysis, the culture medium of the cellscultured on the 6-well plate was removed and washed with DPBS. Ahybrid-R kit (Gene All) was used for extracting RNA, and extracted RNAwas quantified using a nano drop (MICROP UV-Vis Spectrophotometer M600).RNA sequencing analysis was performed by requesting 1 g of RNA toMacrogen.

As a result, it was confirmed that expression of mRNA related tosynthesis of an extracellular matrix and cell interactions involved incell migration and induction of cell aggregates in keratin-treated humandermal papilla cells was greatly increased, and mRNA expressionassociated with cell proliferation was inhibited (FIG. 9).

Further, the human dermal papilla cells were inoculated on a 6-wellplate by 2×10⁵ cells, and after 24 hours, the medium was replaced with aDMEM normal medium at a high concentration or a DMEM normal mediumcontaining 1% (w/v) of keratin at a high concentration. After 3 and 5days of culture, the expression level of the factors for inducing hairgrowth was measured by immunochemical staining.

After the human dermal papilla cells were cultured as described above,the medium was removed and the cells were washed with DPBS. The cellswere immobilized by treating 4% paraformaldehyde for 10 minutes and thenwashed again with DPBS. After immobilization, 0.1% triton X-100 wastreated for 30 minutes to be permeabilized and 10% (w/v) normal goatserum (NGS) was treated for 1 hour. Thereafter, a rabbit anti-humanbeta-catenin antibody diluted at 1:200, a mouse anti-human FGF7antibody, rabbit anti-human FGF7 antibody, a rabbit anti-human FGF10antibody, and a rabbit anti-human BMP6 antibody were treated and reactedat 4° C. for 24 hours. After 24 hours of reaction, the cells were washedthree times with DPBS, and a secondary Alexa Fluor 546 conjugatedantibody and a secondary Alexa Fluor 488-conjugated antibody weretreated at room temperature for 1 hour. Thereafter, the cells werewashed three times with DPBS and treated with4′,6-diamidino-2-phenylindole (DAPI). The stained cells were thenobserved with a fluorescence microscope (Olympus I ×71). As a result,formation of cell aggregates was induced in keratin-treated human dermalpapilla cells, and the molecular expression of FGF7, FGF10 and BMP6,which were known as factors inducing hair growth, was greatly increased(FIG. 10).

Experimental Example 4: Expression Induction of Hair Growth-RelatedFactors of Keratin for Human Outer Root Sheath (ORS) In Vitro

In order to confirm a hair regeneration effect of keratin, expression offactors related to hair growth and induction of hair growth wasconfirmed.

It was confirmed whether a keratin solution prepared using human outerroot sheath cells showed the expression of factors associated with hairgrowth and hair growth induction.

Specifically, the human outer root sheath cells were inoculated on a6-well plate by 5×10⁴ cells, and after 24 hours, the medium was replacedwith an ORS medium or a 1% (w/v) keratin-containing ORS medium. After 3days of incubation, the expression levels of beta-catenin, CD34,P-cadherin, and E-cadherin and the expression levels for transcriptionfactors were measured by immunochemical staining and RT-PCR analysis.

After the human outer root sheath (ORS) cells were cultured as describedabove, the medium was removed and the cells were washed with DPBS. Thecells were immobilized by treating 4% paraformaldehyde for 10 minutesand then washed again with DPBS. After immobilization, 0.1% triton X-100was treated for 30 minutes to be permeabilized and 10% (w/v) normal goatserum (NGS) was treated for 1 hour. Thereafter, a rabbit anti-humanbeta-catenin antibody diluted at 1:200, a rabbit anti-human CD34antibody, a rabbit anti-human P-cadherin antibody, a rabbit anti-humanE-cadherin antibody, and a mouse anti-human integrin beta1 antibody weretreated and reacted at 4° C. for 24 hours. After 24 hours of reaction,the cells were washed three times with DPBS, and a secondary Alexa Fluor546 conjugated antibody, a secondary Alexa Fluor 488-conjugatedantibody, and a PE-conjugated Palloidin antibody were treated at roomtemperature for 1 hour. Thereafter, the cells were washed three timeswith DPBS and treated with 4′,6-diamidino-2-phenylindole (DAPI). Thestained cells were then observed with a fluorescence microscope (OlympusI ×71). For RT-PCR analysis, the culture medium of the cells cultured inthe 6-well plate was removed and the cells were washed with DPBS. Ahybrid-R kit (Gene All) was used for extracting RNA, and extracted RNAwas quantified using a nano drop (MICROP UV-Vis Spectrophotometer M600).1 g of RNA was added to AccuPower Cycle Script RT Premix (Bioneer,Daejeon, Korea) and cDNA was prepared using a thermal cycler (T106,Bio-Rad). The sequences of primers used are illustrated in Table 2below.

TABLE 2 Sense(5′-3′) Antisense(5′-3′) beta- TGCAGTTCGCCTTCACTATGCTGCACAAACAATGGAATGG catenin (SEQ ID NO: 1) (SEQ ID NO: 2) KRT5ACCAGTACCCGCATCTGCA TGTTCCGTGGCCTCTTCG (SEQ ID NO: 3) (SEQ ID NO: 4)MSX2 CCGCCAAGACATATGAGCCC ACCTGGGTCTCTGTGAGGTT (SEQ ID NO: 5)(SEQ ID NO: 6) ITGA6 AGCTGTGCTTGCTCTACCTG CCGGGGTCTCCATATTTCCG(SEQ ID NO: 7) (SEQ ID NO: 8) FOXN1 AGTGGTGCTGGGATGTTCTGATAGTGTGAGGAGCCCAGGT (SEQ ID NO: 9) (SEQ ID NO: 10) CD10CTTTAGTGCCCAGCAGTCCA GAGTCCACCAGTCAACGAGG (SEQ ID NO: 11)(SEQ ID NO: 12) beta- GTCAGGCAGCTCGTAGCTCT TCGTGCGTGACATTAAGGAG actin(SEQ ID NO: 13) (SEQ ID NO: 14)

To each reaction, 10 μl of SYBR Green PCR Mix, 0.5 pm of primers (senseand antisense) and 50 nm of a template were added. RT-PCR was performedusing RG6000 5plex HRM (Corbett Research) instrument and 40 cycles wereset at 95° C. for 15 seconds and annealing temperature of 57° C. for 45seconds. A comparative quantification (Ct(2-^(ΔΔCt))) method was used tomeasure a threshold cycle (Ct) value.

As a result, in the case of the human outer root sheath (ORS) cells, inthe treatment of keratin, it was confirmed that the expression ofbeta-catenin and P-cadherin, which were factors involved in migration ofouter root sheath cells and differentiation into matrix cells inrelation with hair regeneration, was increased, and the expression ofCD34 known as a molecule of which expression was decreased indifferentiation was reduced (FIG. 11).

In addition, it was confirmed that the expression of MSX2, FOXN1 andCD10 genes associated with the differentiation of human outer rootsheath (ORS) cells into matrix cells during keratin treatment wasincreased (FIG. 12), and the expression of KRT5 and ITGA6 genes known tobe decreased in expression upon differentiation was decreased (FIG. 12).

Experimental Example 5: Expression Induction of Hair Growth-RelatedFactors of Hydrolyzed Keratin for Human Outer Root Sheath Cells In Vitro

In order to confirm a hair regeneration effect of hydrolyzed keratin,the expression of factors related to hair growth and stemness wasconfirmed.

The human outer root sheath (ORS) cells were inoculated on a 6-wellplate by 5×10⁴ cells, and after 24 hours, the medium was replaced withan ORS medium or a 1% (w/v) hydrolyzed keratin-containing ORS medium.After 1, 3 and 5 days of culture, expression levels of beta-catenin,Sox-9, and CD44 were measured by immunochemical staining and RT-PCRanalysis.

After the human outer root sheath (ORS) cells were cultured as describedabove, the medium was removed and the cells were washed with DPBS. Thecells were immobilized by treating 4% paraformaldehyde for 10 minutesand then washed again with DPBS. After immobilization, 0.1% triton X-100was treated for 30 minutes to be permeabilized and 10% (w/v) normal goatserum (NGS) was treated for 1 hour. Thereafter, a rabbit anti-humanbeta-catenin antibody diluted at 1:200, a rabbit anti-human Sox-9antibody, and a rabbit anti-human CD44 antibody were treated and reactedat 4° C. for 24 hours. After 24 hours of reaction, the cells were washedthree times with DPBS, and a secondary Alexa Fluor 546 conjugatedantibody and a secondary Alexa Fluor 488-conjugated antibody weretreated at room temperature for 1 hour. Thereafter, the cells werewashed three times with DPBS and treated with4′,6-diamidino-2-phenylindole (DAPI). The stained cells were thenobserved with a fluorescence microscope (Olympus I ×71). For RT-PCRanalysis, the culture medium of the cells cultured in the 6-well platewas removed and the cells were washed with DPBS. A hybrid-R kit (GeneAll) was used for extracting RNA, and extracted RNA was quantified usinga nano drop (MICROP UV-Vis Spectrophotometer M600). 1 g of RNA was addedto AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Korea) and cDNAwas prepared using a thermal cycler (T106, Bio-Rad). The sequences ofprimers used are illustrated in Table 3 below.

TABLE 3 Sense(5′-3′) Antisense(5′-3′) beta- TGCAGTTCGCCTTCACTATGCTGCACAAACAATGGAATGG catenin (SEQ ID NO: 1) (SEQ ID NO: 2) Sox-9ACCAGTACCCGCATCTGCA TGTTCCGTGGCCTCTTCG (SEQ ID NO: 15) (SEQ ID NO: 16)beta- GTCAGGCAGCTCGTAGCTCT TCGTGCGTGACATTAAGGAG actin (SEQ ID NO: 13)(SEQ ID NO: 14)

To each reaction, 10 μl of SYBR Green PCR Mix, 0.5 pm of primers (senseand antisense) and 50 nm of a template were added.

RT-PCR was performed using RG6000 5plex HRM (Corbett Research)instrument and 40 cycles were set at 95° C. for 15 seconds and annealingtemperature of 57° C. for 45 seconds. A comparative quantification(Ct(2-^(ΔΔCt))) method was used to measure a threshold cycle (Ct) value.

After the human outer root sheath (ORS) cells were cultured as describedabove, the medium was removed and the cells were washed with DPBS. 0.3to 1 ml of a protein lysis buffer (Cell Lysis Buffer (10×): RiPA buffer(biorad-89901) and Phosphatase Inhibitor Cocktail (thermofisher (100×),78440) was added and cells were collected with a scraper and transferredto an e-tube. A protein-containing supernatant was obtained bycentrifuging the cells at 4° C. for 30 minutes using a centrifuge(Centrifuge 5427 R, effendorf 10,000×g). Protein quantification wasperformed using a Bio Rad Protein Assay reagent. 30 μg of a proteinsample was added with a 6× sample dye (DTT, sigma 43815, bromophenolblue sigma, BR0222, Glycerl, bioshop, GLY001) and mixed to become 1×,and the sample was added to a heating block (HYSC_Introduction_HeatingBlock_2014) and heated at 95° C. for 10 minutes. PROTEAN® Tetra HandcastSystems (Bio-rad) were prepared by lightly spinning-down and vortexing(Stuart Scientific SA8 vortex mixer AC input 90/230 V, 50-60 Hz) thesample, and then leaving the sample in the ice. In order to prepare agel, short glass and long glass were assembled in plates for handcasting, and a glass plate was washed with 70% ethanol (merk) anddistilled water and then put in a frame, and a grease was applied to thebottom of the plate which hardened the gel to prevent the gel fromleaking. A final 22 ml of separating gel (containing 6 ml of 30%acrylamide (30% Acrylamide/Bis Solution, 19:1 #1610154), 5 ml of dH₂O,3.75 ml of 1.5 M Tris-HCl (Sigma, T1503), 150 μl of SDS (sigma, sodiumdodecyl sulfite, L3771), 7.5 μl of TEMED (USB, 76230), and 75 μl of 10%ammonium persulfate (sigma, 215589)) was prepared and placed in a glassplate, and then added with 70% ethanol to be leveled. After about 30minutes, the glass plate was tilted to remove ethanol, and then a final10 ml of stacking gel (containing 1.3 ml of 30% acrylamide (30%Acrylamide/Bis Solution, 19:1 #1610154), 6.1 ml of dH₂O, 2.5 ml of 1.0 MTris-HCl (Sigma, T1503), 100 μl of HCl (sigma, T1503), 10 μl of TEMED(USB, 76230), and 60 μl of 10% ammonium persulfate (sigma, 215589)) wasprepared. The stacking gel was added, a 1.0 mm comb was inserted, andthen the gel plate was set in an electrophoretic tank. A sample wasloaded into each well and 5 μl of a size marker (ladder) (proteinmolecular weight marker 10 to 245 kDa ab116028-Abcam) was also added. Amini gel was reacted at 50V for 20 minutes and electrophoresed for 1hour to 1.5 hours while increasing to 100V when passing through thestacking gel. After the electrophoresis was completed, the gel wasseparated from the gel plate and placed in a transfer buffer. Meanwhile,the transfer buffer was prepared using 14.4 g of Glycine (Sigma), 3 g ofTrizma base (Sigma, T1503), 200 mL of methanol, and 800 mL of dH₂O. APVDF transfer membrane (millipore cat No ISEQ 10100 10 cm×10 cm PVDF)was immersed in methanol for 5 minutes, in tertiary H₂O for 3 minutesand in a transfer buffer for 3 minutes and shaken, and then a cassettesandwich was made in the following order: (-) Black plate—Sponge—3Mpaper—Transparent plate transfer device (Bio-rad). The cassette wasplaced in a water bath and transferred at 200 V for 90 minutes on astirrer. After the transfer was completed, the band was confirmed bydying the gel and the membrane. 2.5 g of ponceau S and 5 ml of aceticacid were added to dH₂O to prepare a reagent solution having a volume of500 ml, and then the entire membrane was shaken in the reagent solutionfor about 5 minutes and then washed with H₂O 2 to 3 times, and as aresult, the band was confirmed and the ladder size was displayed. Ablocking solution was prepared with 10 g of skim milk (BD, 2322100) and200 ml of TBS (Tris 12 g, NaCl 9 g in dH₂O), the membrane was placed ina plastic bowl, and then cells were incubated in 10 ml of the blockingsolution for 1 hour. A B-catenin primary antibody (B-catenin antibody[E247] (ab32572)) and a B-Actin primary antibody (β-Actin Antibody (C4))diluted at 1:1000 were added to 10 ml of the blocking solution andincubated in a Santa Cruz incubator at 4° C. for 24 hours. The cellswere washed three times for 5 min each with TBST (Tris 12 g, NaCl 9 g indH₂O, 1% tween 20) and a B-catenin secondary antibody (goat anti-rabbitIgG-HRP: sc-2004) and a B-Actin secondary antibody (mouse anti-rabbitIgG-HRP) diluted at 1:10000 were incubated in 10 ml of the blockingsolution for 2 hours. The cells were washed three times for 5 min eachwith TBST (Tris 12 g, NaCl 9 g in dH₂O, 1% tween 20) and then ECLSubstrate (Daemyung Science) was added and incubated for 5 minutes. In adark room, a membrane was placed on a development cassette (DaemyungScience, High-Sensitivity Screen), an X-ray film (Daemyung Science 8×10inch) was incubated for 5 minutes, and the film was taken out anddeveloped on an automatic developing machine (SAEKI, P & C, ATL-1500).

As a result, it was confirmed that in the case of human outer rootsheath (ORS) cells, the expression of a beta-catenin molecule which is afactor related with migration of outer root sheath cells and activity ofclone and a Sox-9 molecule involved in stemness was increased inrelation with hair regeneration in the treatment of hydrolyzed keratin(FIG. 13). In addition, it was confirmed that the expression of a Sox-9gene and a beta-catenin gene involved in stemness was increased (FIG.14). In particular, it was confirmed that beta-catenin was expressedfour times or more when treated with hydrolyzed keratin (FIG. 15).

Experimental Example 6: Expression Induction of Hair Growth-RelatedFactors of Hydrolyzed Keratin for Human Dermal Papilla Cells In Vitro

Human dermal papilla (DP) cells were inoculated by 2×10⁵ cells in a6-well plate without any treatment. After 24 hours, the medium wasreplaced with a DP medium or a DP medium containing 1% (w/v) ofhydrolyzed keratin. After 1, 2, and 3 days of incubation, the formationdegree of cell aggregates and the activity degree of alkalinephosphatase were analyzed, and expression levels of beta-catenin, Sox-2,CD133, alkaline phosphatase, FGF7, and FGF10 were measured throughimmunohistochemical staining.

During the culturing of dermal papilla (DP) cells as described above,the number of cell aggregates produced during the treatment ofhydrolyzed keratin was measured by an optical microscope.

In addition, after the culturing of dermal papilla (DP) cells asdescribed above, the cells were immobilized by treating 4%paraformaldehyde for 1 to 2 minutes, washed with a rinse buffercontained in an alkaline phosphatase detection kit (Merk MilliporeSCR004), added with 1 ml of a reaction solution in which Naphthol AS-BIphosphate solution: Fast Red Violet solution: distilled were mixed at2:1:1, and then reacted at room temperature under a dark condition for15 minutes. After the reaction, the cells stained by the activity of thealkaline phosphatase expressed by the alkaline phosphate cells wereobserved with a fluorescence microscope (Olympus I ×71).

After the human dermal papilla (DP) cells were cultured as describedabove, the medium was removed and the cells were washed with DPBS. Thecells were immobilized by treating 4% paraformaldehyde for 10 minutesand then washed again with DPBS. After immobilization, 0.1% triton X-100was treated for 30 minutes to be permeabilized and 10% (w/v) normal goatserum was treated for 1 hour. Thereafter, a rabbit anti-humanbeta-catenin antibody diluted at 1:200, a mouse anti-human Sox-2antibody, a mouse anti-human ALPase antibody, a rabbit anti-human CD133antibody, a mouse anti-human FGF-7 antibody, and a rabbit anti-humanFGF-10 antibody were treated and reacted for 4° C. for 24 hours. After24 hours of reaction, the cells were washed three times with DPBS, and asecondary Alexa Fluor 546 conjugated antibody and a secondary AlexaFluor 488-conjugated antibody were treated at room temperature for 1hour. Thereafter, the cells were washed three times with DPBS andtreated with 4′,6-diamidino-2-phenylindole (DAPI). The stained cellswere observed with a fluorescence microscope (Olympus I ×71).

As a result, it was confirmed that in the case of dermal papilla (DP)cells, in the treatment of hydrolyzed keratin, the formation of cellaggregates involved in hair regeneration of dermal papilla cells andstemness was increased 5 times or more (FIG. 16), and the activity ofthe alkaline phosphatase expressed by alkaline phosphate cells wasincreased (FIG. 17). Furthermore, it was confirmed that the expressionof beta-catenin, Sox-2, alkaline phosphatase, CD133, FGF-7, and FGF-10molecules as factors for the hair regeneration of dermal papilla cellsand stemness was increased (FIG. 18).

Through Experimental Examples 1 and 6, it was found that keratinincreased the stemness of stem cells involved in hair regeneration andinduced cell migration and differentiation to generate cell aggregates,thereby effectively inducing regeneration of hair.

1-13. (canceled)
 14. An injectable pharmaceutical composition comprisingkeratin for stimulating hair growth and regenerating dermal papilla,wherein the keratin has a molecular weight of 500 to 70,000 Daltons bydisulfide bond degradation or peptide degradation.
 15. The injectablepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition increases an expression level of at least one selected fromthe group consisting of beta-catenin, Sox-9, Sox-2, alkalinephosphatase, CD133, FGF7, FGF10, BMP6, P-cadherin, E-cadherin, MSX2,FOXN1 and CD10.
 16. The injectable pharmaceutical composition of claim1, wherein the pharmaceutical composition is administered to the dermallayer or the subcutaneous tissue.
 17. The injectable pharmaceuticalcomposition of claim 1, further comprising: a penetration enhancer. 18.The injectable pharmaceutical composition of claim 8, wherein thepenetration enhancer is at least one selected from the group consistingof sulphoxide, azone, pyrrolidone, fatty acids, lower alcohols having 1to 4 carbon atoms, higher fatty alcohols having 6 or more carbon atoms,glycols, urea, terpene, terpenoid, and phospholipid.
 19. The injectablepharmaceutical composition of claim 1, wherein the keratin is thekeratin by the peptide degradation prepared by a preparation method,including S1) reacting keratin with hydrolase; and S2) removing thehydrolase.
 20. The injectable pharmaceutical composition of claim 10,wherein the hydrolase is at least one selected from the group consistingof Proteinase-K, Leucyl aminopeptidase, carboxypeptidase, pepsin,trypsin, and chymotrypsin.
 21. The injectable pharmaceutical compositionof claim 10, wherein the hydrolase is immobilized on beads.
 22. Theinjectable pharmaceutical composition of claim 10, wherein thepreparation method further includes S3) removing the activity of thehydrolase after step S2).